Expression of constitutively active M-Ras G22V in an IL-3–dependent cell line results in factor-independent growth. (A) Polyclonal, puromycin-resistant R6-X M-Ras G22V cells (□) or parental R6-X cells (▪) were plated in agar in triplicate in the absence of IL-3 at 2.5 × 104 per mL or 2.5 × 103/mL (as indicated). Colonies were counted at day 7 using a colony-microscope. (B) Correlation of levels of expression of M-Ras G22V with factor-independent growth. Clones of R6-X M-Ras G22V cells derived from colonies grown in medium alone (M series; ⧫) or in the presence of IL-3 (F series; □) were expanded in IL-3, washed, and incubated in medium alone for days, when survival and growth were assessed by uptake of MTT as described above. The uptake of MTT (expressed as OD units) was plotted against the mean intensity of fluorescence (MFI) of EGFP assessed by flow cytometry of the same clones grown in the presence of IL-3. (C) Correlated expression of EGFP and M-Ras G22V. Twenty micrograms of whole-cell lysates of 4 of the clones shown in (B) above, 2 exhibiting high EGFP expression (M.4, MFI 1775, and M.6, MFI 2271) and 2 with low EGFP expression (F.1, MFI 216, and F.3, MFI 474), were immunoblotted using a monoclonal antibody specific for the HA-tag present on the M-Ras G22V. Also shown are lysates of parental R6-X cells and of a “sorted,” polyclonal population of M-Ras G22V-expressing R6-X cells, selected by fluorescence-activated cell sorting (FACS) and culture in IL-4 (sorted), and of a population of R6-X cells infected with empty vector (vector alone).
